Fig. 3: MyoD directly instructs chromatin looping dependent on the E-box.

a MyoD mediates chromatin looping in vitro. The upper panel presents a schematic for the in vitro DNA circularization assay used to detect the ability of MyoD (green) to mediate DNA looping with E-box (red)-containing DNA (3.8 kb) and the no-E-box control (2.7 kb). The lower panel presents representative views of circularized DNA mediated by recombinant MyoD protein, as imaged by transmission electron microscopy (TEM). The images are representative of three independent experiments. b Hi-C contact map depicting normalized contact frequencies and ChIP-seq signal tracks for a MyoD-bound chromatin loop at the MyoG locus. The examined chromatin loop, which shows reduced Hi-C contacts in the MKO-DM sample compared with the WT-DM sample, is marked by a yellow circle. c Schematic showing CRISPR/Cas9-mediated deletion of the MyoD E-box in the regulatory region of the MyoG locus. d Representative images of 3D-DNA fluorescent in situ hybridization (3D-DNA FISH) in WT and E-box-deleted cells (described in c) cultured in DM. The upper panel shows that the MyoG promoter (red) and Mybph promoter (green) are separated by more in the E-box-deleted cells than in the WT cells. The lower panel showed the distance between the MyoG-Mybph loop anchors. The scale bar represents 5 μm. Data are presented as mean ± SEM. p-values were determined using unpaired two-tailed Student’s t-test, ***p < 0.001. Source data are provided as a Source Data file. e Representative dCas9-mediated DNA imaging (upper panel), which was used to visualize the chromatin loop anchors described in b. The distances between the two anchors were measured (lower panel). MKO cells were infected with adenovirus expressing MyoD (Ad-MyoD) or Ad-EGFP (control, Ad-Ctrl). The chromatin loopings at MyoG (red) and Mybph (green) loci were visualized by dCas9-mediated DNA imaging. DAPI served to visualize nuclei. The scale bar represents 5 μm. Data are presented as mean ± SEM. p-values were determined by performing ANOVA followed by Tukey’s multiple comparison test, ***p < 0.001. Source data are provided as a Source Data file.